Standard Method

STANDARD PLATE COUNTING PROCEDURE

For Determining The Viable Count In Enzyme/Culture Products

For ascertaining the viable cell count of the various crude fermentation products/culture
concentrates that are manufactured for use in feed and waste treatment formulations, the following adapt ions of the APHA Standard methods procedure (Section 406) is used.

It is important that the initial soaking/dispersion step as outlined below be followed in order to release immobilized bacterial cells in these types of products and thus enable the counting procedure to yield consistent and valid results. Other soaking or dispersion techniques may not give comparable results with these products. The initial dilution blank of, 50 - 100 ml of sterile physiological saline solution in a 125 - 250 ml Erlenmeyer flask should contain approximately 15 grams of 5 - 7 mm glass beads which had been added to the blank prior to sterilization. At least 2.0 grams of dry product to be counted should be emptied into the initial blank which contains the glass bead sterile saline and is maintained at a
temperature of 25 - 35° C. The material is allowed to soak for 5 minutes. At the end of the five minutes soaking period, the flask is
covered with a a sterile neoprene stopper of proper size and the flask should be agitated with up and down motion for a period of five minutes, at the rate of one
oscillation or arc per second, each arc being approximately of one foot magnitude.

At the end of the five minutes soaking and five minutes initial disruption period, further serial dilution of the product is accomplished in such a manner that upon final plating that colonies numbering between 30 and 300 per plate occur in at least one level of dilution magnitude. plating should be executed in triplicate. Pour plating technique should be used. Plates are incubated for 48 hours
@ 35° C. They should be examined at 24, 30 and 48 hours for proper amount of growth to allow counting.

Care should be taken to see that plates are dry before incubation. Some bacilli will quickly spread on a damp plate and completely cover a
plates surface, making counting very difficult.

FOR AEROBIC
COUNTS:

Agar plates are incubated for an appropriate length of time (as specified above) in regular incubator under aerobic conditions.

FOR ANAEROBIC
COUNTS:

Agar plates are first placed into a suitable anaerobic jar or vessel such as that manufactured by Oxoid or Gaspak. The
anaerobic system should have a resazurin indicator (which indicates the condition of the reduced environment). The anaerobic system should be incubated under the
same conditions as outlined above.

FOR SPORE
COUNTS:

In addition to the dilution steps outlined above, 1.0 m1. of final dilution blank is withdrawn aseptically and placed into a sterile 10
mI. screw cap tube. The tube is immersed halfway in a water bath maintained at
80° C +/- 0.5° C for a period of 5 minutes. The tube is then withdrawn and placed in an ice bath to cool. After cooling for 5 minutes in the ice bath, an appropriate amount of dilution blank is withdrawn with a sterile volumetric pipette and plated as above. The plates are incubated in the same manner as
"FOR AEROBIC COUNTS" above.